Phage and use thereof in soil remediation

Abstract

A phage and use thereof in soil remediation are disclosed. The phage φYSZPK has been deposited at the China Center for Type Culture Collection on Aug. 1, 2018 under Accession No. CCTCC M 2018516, and its taxonomic designation is Pseudomonas aeruginosa and Klebsiella phage φYSZPK. Biochar and the screened phage are combined and returned into contaminated soil to synergistically control and deeply track and inactivate transmission and spread of antibiotic resistance pathogenic bacteria and resistance genes in a soil-vegetable system. The combination of the biochar and the phage φYSZPK not only clearly improves the functional stability of microbial community in the soil-vegetable system, but also significantly alleviates the dissemination of the antibiotic resistance pathogenic bacteria in the soil-vegetable system to prevent secondary pollution, thereby providing a new solution for biological remediation and control of farmland soil contaminated by antibiotic resistance pathogenic bacteria in China.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This U.S. application claims benefit and priority to Chinese Application No. 201810959953.9 filed on Aug. 22, 2018, which is incorporated by reference herein in its entirety.FIELD OF THE INVENTION[0002]The present invention belongs to the technical field of remediation of compound contaminated soil of multiple antibiotic resistance pathogenic bacteria and related resistance genes thereof, and particularly relates to a phage φYSZPK and use thereof in controlling and inactivating antibiotic resistance pathogenic bacteria in a soil-vegetable system.DESCRIPTION OF RELATED ART[0003]In recent years, due to the abuse of antibiotic veterinary drugs, the defect of a safe treatment technology for livestock and poultry manure, and the lack of environmental management, in China and many countries around the world, farmland soil-vegetable systems around suburban livestock farming plants often become high-risk hotspot “sources” and “sinks” for residues...

AI Technical Summary

Benefits of technology

[0020]The working principle of the present invention is as follows: 1. the biochar is a kind of environment-friendly material which is more in porosity, high in specific surface area, stable in chemical structure and rich in surface functional groups and contains a large number of elements such as C, N, P, Ca, and Mg; 2. the biochar can adsorb and control contaminants (including pesticide residues, organic solvents, heavy metal ions, polycyclic aromatic hydrocarbons, and the like) in soil or water so as to reduce the environmental risk, and furthermore, the biochar can be applied into the soil to achieve the effects of preserving moisture and fertility, improving physical and chemical properties of the soil and increasing the crop yield; 3. phages are a kind of bacterial viruses surviving by specifically infecting host bacteria, and can be divided into lytic phages and lysogenic phages; 4. the lytic phages can recognize the specific binding sites of the surfaces of cell membranes of host bacteria in the environmental migration process and perform paired adsorption, subsequently, the tail sheaths of the phages shrink, the DNA of the nucleic acids is injected into the host bacteria through hollow tails to execute an invasion process, then, the DNA of the phages utilizes the nucleic acid base pairs and energy substances in the host to rapidly complete its own nucleic acid replication and protein synthesis, a large number of progeny phages are assembled and proliferated in the bacteria, and cell wall lytic enzymes are released, thereby causing the host bacteria to rupture and die and destroying the internal structure of the bacteria so as to finally complete lysis and release processes; 5. a lytic phage with high titer, short lysis period and strong stress resistance is selected by simulation according to in-situ contaminated soil environmental conditions (temperature, pH, ion concentration, and the like) so as to be used as a preferred strain for phage therapy, the lytic phage will preferentially “prey on” pathogenic bacteria with higher specificity in the environment, after this type of pathogenic bacteria are reduced to a certain level, the lytic phage will attack host bacteria with weaker specificity and maintain its survival state at a certain level, and furthermore, secondary pollution of the pathogenic bacteria can be prevented; 6. there are ten or dozens of phages around each bacterium in the soil environment, and some phages will be transferred into vegetables by means of plant root penetration and leaf surface transpiration to simultaneously track and inactivate resistance pathogenic bacteria in the vegetables, thereby controlling the transmission and spread of the resistance pathogenic bacteria and indirectly preventing the human health from being affected due to food chain transfer action; 7. the selected phages are derived from the soil and finally returned to the soil without any modification, and thus are environmentally friendly; 8. the ecological risk after application of the phage therapy is evaluated to ensure the ecological functional diversity and stability of microorganisms.

[0021]The invention relates to a method for controlling and inactivating compound high-abundance resistance pathogenic bacteria in soil-vegetable systems by combination of biochar and phage therapy, and provides a rapid remediation technology. The present invention has the following main advantages: 1. The biochar can broadly control compound resistance pathogenic bacteria and resistance genes in the contaminated soil; 2. The application of the biochar is favorable for preserving moisture and fertility of soil, improving the crop quality and increasing the crop yield; 3. The phage can realize targeted inactivation of resistance pathogenic bacteria in the contaminated soil, and can simultaneously reduce the abundance of related resistance genes; 4. The biochar and the phage are low in preparation cost, convenient in storage and transport and simple and convenient in use and operation, can realize broad spectrum control and accurate targeted inactivation, can prevent secondary pollution after remediation, and are easy for popularization; 5. The combination of the biochar and the phage can clearly improve the ecological functional diversity and stability of microorganisms in soil, and is environmentally friendly. The method has broad application prospects for remediation of compound high-concentration antibiotic resistance pathogenic bacterium contaminated soil around a large number of livestock and poultry farms in China.

Problems solved by technology

In recent years, due to the abuse of antibiotic veterinary drugs, the defect of a safe treatment technology for livestock and poultry manure, and the lack of environmental management, in China and many countries around the world, farmland soil-vegetable systems around suburban livestock farming plants often become high-risk hotspot “sources” and “sinks” for residues and breeding of antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs).

Especially, under the promotion of horizontal transfer or vertical transduction of a large number of movable gene elements (plasmids, integrons, and transposons) in an environment, the risk of spread and transmission of some zoonotic antibiotic resistance pathogenic bacteria is greatly increased, and furthermore, it causes a very serious potential threat to human health and ecological safety.

However, there is almost no remediation technology for combination of the biochar and the phage therapy to reduce the compound contamination of ARB and ARGs in soil-vegetable systems.

These methods all indicate that both the biochar and the phage can control and even inactivate pathogenic bacteria, and can simultaneously prevent diseases of crops, but generally lack the overall remediation effect on synergistic removal of ARB and ARGs in soil-crop (vegetable) systems.

However, there are fewer researches on broad-spectrum adsorption and removal of antibiotic resistance pathogenic bacteria in soil.

Furthermore, the related ecological risk assessment of the functional stability and diversity of microbial community in soil-vegetable systems after inoculation is relatively scarce.

In most of the existing phage therapy applications, phages with strong selectivity and high specificity are used as primary materials, biological control technologies for simultaneous inactivation of the compound contamination of multiple antibiotic resistance pathogenic bacteria in soil-vegetable systems are lacking, and furthermore, the related ecological risk assessment of the functional stability and diversity of microbial community in soil-vegetable systems after inoculation has received little attention.

The main reasons for the defects are as follows: In recent years, the academic community has gradually recognized that soil-vegetable systems are “sources” and “sinks” for accumulation of multiple antibiotic resistance bacteria and resistance genes, and this type of novel resistance pathogenic bacteria and genes will seriously threaten the human health and ecological environment safety through the transfer action of food chains.

Images